Dual-color visualization of trans-Golgi network to plasma membrane traffic along microtubules in living cells

Abstract

The mechanisms and carriers responsible for exocytic protein trafficking between the trans-Golgi network (TGN) and the plasma membrane remain unclear. To investigate the dynamics of TGN-to-plasma membrane traffic and role of the cytoskeleton in these processes we transfected cells with a GFP-fusion protein, vesicular stomatitis virus G protein tagged with GFP (VSVG3-GFP). After using temperature shifts to block VSVG3-GFP in the endoplasmic reticulum and subsequently accumulate it in the TGN, dynamics of TGN-to-plasma membrane transport were visualized in real time by confocal and video microscopy. Both small vesicles (<250 nm) and larger vesicular-tubular structures (>1.5 microm long) are used as transport containers (TCs). These TCs rapidly moved out of the Golgi along curvilinear paths with average speeds of approximately 0.7 micrometer/second. Automatic computer tracking objectively determined the dynamics of different carriers. Fission and fusion of TCs were observed, suggesting that these late exocytic processes are highly interactive. To directly determine the role of microtubules in post-Golgi traffic, rhodamine-tubulin was microinjected and both labeled cargo and microtubules were simultaneously visualized in living cells. These studies demonstrated that exocytic cargo moves along microtubule tracks and reveals that carriers are capable of switching between tracks.